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1.
Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication 总被引:24,自引:0,他引:24
Mostafa Motallebi-Veshareh † Dietmar Balzer Erich Lanka Grazyna Jagura-Burdzy Christopher M. Thomas 《Molecular microbiology》1992,6(7):907-920
The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid. 相似文献
2.
Grazyna Adamus Z. Suzanne Zam Scotts Emerson Paul A. Hargrave 《In vitro cellular & developmental biology. Plant》1989,25(12):1141-1146
Summary We present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired
cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower
than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the
chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate
the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting
to isolate a hydridoma cell line that is relatively rare in a population.
This work was supported in part by grants EY 06225 and EY 06226 from the National Eye Institute of the National Institutes
of Health, Bethesda, MD and by an unrestricted departmental award from Research to Prevent Blindness, Inc. 相似文献
3.
Abstract Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450(cam). The effect of the alignment, loop choice, and minimization with or without water was assessed. Although final models were similar in overall structure, the identity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choice of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agreement with the data from site-directed mutagenesis studies, and correctly predicted the effect of substitutions at 9 out of 10 amino acid positions. Thus, residues important for P450 2B1 activity, such as Ile- 114, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part of the active site and are able to interact with the substrate androstenedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interact with the substrate, in agreement with experimental data. The model indicates other residues likely to be important for enzyme function, such as Tyr- 111, Leu-209, Ile-477, and Ile- 480, which can be tested experimentally. The substrate may assume numerous binding orientations consistent with observed patterns of hydroxylation at C(5) and C(6). The replacement in the model of certain amino acid residues to mimic residue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible explanation for alterations in regio- and stereospecificities of some mutants of P450 2B1, such as Gly-478 → Ala or Val-363 Ala. 相似文献
4.
5.
Grazyna Formicka-Kozlowska Leslie D. Pettit Ian Steel Bianka Hartrodt Klaus Neubert Piotr Rekowski Gotfryd Kupryszewski 《Journal of inorganic biochemistry》1984,22(3):155-163
The synthesis of β-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly, H2L) and a number of its peptide fragments is described. Complexes formed between these peptides and Cu(II) have been investigated spectrophotometrically, using CD and EPR spectroscopy, and potentiometrically. Results show that, with tyrosine as the N-terminal residue, the major complex formed at physiological pH is the dimeric species, [Cu2L2], bonded through the phenolic O? of the Tyr residue of one ligand and the N-terminal amine nitrogen of the second ligand molecule. There is no evidence for coordination through the peptide nitrogens unless the terminal Tyr group is removed. 相似文献
6.
Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV. 相似文献
7.
Perlińska-Lenart U Orlowski J Laudy AE Zdebska E Palamarczyk G Kruszewska JS 《Applied and environmental microbiology》2006,72(12):7778-7784
Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene. 相似文献
8.
Grazyna Majkowska-Skrobek Pawel Markwitz Ewelina Sosnowska Cédric Lood Rob Lavigne Zuzanna Drulis-Kawa 《Environmental microbiology》2021,23(12):7723-7740
Bacteriophage therapy is currently being evaluated as a critical complement to traditional antibiotic treatment. However, the emergence of phage resistance is perceived as a major hurdle to the sustainable implementation of this antimicrobial strategy. By combining comprehensive genomics and microbiological assessment, we show that the receptor-modification resistance to capsule-targeting phages involves either escape mutation(s) in the capsule biosynthesis cluster or qualitative changes in exopolysaccharides, converting clones to mucoid variants. These variants introduce cross-resistance to phages specific to the same receptor yet sensitize to phages utilizing alternative ones. The loss/modification of capsule, the main Klebsiella pneumoniae virulence factor, did not dramatically impact population fitness, nor the ability to protect bacteria against the innate immune response. Nevertheless, the introduction of phage drives bacteria to expel multidrug resistance clusters, as observed by the large deletion in K. pneumoniae 77 plasmid containing blaCTX-M, ant(3″), sul2, folA, mph(E)/mph(G) genes. The emerging bacterial resistance to viral infection steers evolution towards desired population attributes and highlights the synergistic potential for combined antibiotic-phage therapy against K. pneumoniae. 相似文献
9.
Joanna Suliburska Pawel Bogdanski Ewa Gajewska Grazyna Kalmus Magdalena Sobieska Wlodzimierz Samborski 《Journal of physiology and biochemistry》2013,69(4):847-853
Some data indicate a potential relationship between insulin resistance level and the concentration of osteoprotegerin (OPG) in the body. There have been few studies concerning OPG level and its relationship with insulin resistance and body composition in young people. The aim of this study was to assess serum osteoprotegerin concentration in obese adolescents, and to evaluate its potential association with insulin resistance. Seventy-eight obese adolescents and 20 healthy volunteers aged 12–18 years were recruited in the study. Selected anthropometrical measurements and blood biochemical analyses were performed. Insulin resistance in the participants was evaluated according to the homeostasis model assessment-insulin resistance (HOMA-IR) protocol. Level of OPG was assessed in serum. Obese subjects had significantly higher HOMA-IR indices and OPG levels in serum than the control group. A significant positive correlation between OPG and insulin resistance was found. It was observed that high concentrations of osteoprotegerin are associated with insulin resistance in obese adolescents. 相似文献
10.
Tomasz Poplawski Cezary Chojnacki Anna Czubatka Grazyna Klupinska Jan Chojnacki Janusz Blasiak 《Molecular biology reports》2013,40(8):5205-5212
Helicobacter pylori (H. pylori) infection plays an important role in gastric carcinogenesis. This bacterium may induce cancer transformation and change the susceptibility of gastric mucosa cells to various exogenous dietary irritants. The aim of the study was to evaluate the influence of H. pylori infection on the reaction of the stomach cells to a genotoxic effect of heterocyclic amines (HCAs). These well-known mutagens are formed during cooking of protein-rich foods, primarily meat. Taking into account that persons consuming a mixed-western diet are exposed to these compound nearly an entire lifetime and more than half of human population is infected with H. pylori, it is important to assess the combined effect of H. pylori infection and HCAs in the context of DNA damage in gastric mucosa cells, which is a prerequisite to cancer transformation. We employed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) because these substances are present in a great amount in cooked and fried meat. Using alkaline comet assay, we showed that the extent of the DNA damage induced by HCAs was significantly higher in H. pylori infected gastric mucosa cells than in non-infected counterparts. We did not observed any difference in the efficiency of repair of DNA lesions induced by HCAs in both type of cells. Vitamin C reduced the genotoxic effects of HCAs in H. pylori infected and non-infected gastric mucosa cells. Melatonin more effectively decreased DNA damage caused by HCAs in H. pylori infected gastric mucosa cells as compared with control. Our results suggest that H. pylori infection may influence the susceptibility of gastric mucosa cells to HCAs and dietary antioxidative substances, including vitamin C and melatonin may inhibit the genotoxic effects of HCAs on gastric mucosa cells and may reduce the risk of carcinogenesis caused by food borne mutagens and H. pylori infection. 相似文献